Optimization of RNAi efficiency in PVD neuron of C. elegans

PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to its elaborate and stereotype dendrite morphology. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans. However, the feeding method often fails when it comes to knocking down genes in nervous system. In order to optimize the RNAi conditions for PVD neuron, we fed the worm strains with E. coli HT115 bacteria expressing dsRNA against mec-3, hpo-30, and tiam-1, whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the available sensitive backgrounds including the one expresses sid-1 under unc-119 promoter, although resulted in reduction of dendrite branching, the phenotypes were significantly modest compared to the respective loss of function mutants. In order to enhance RNAi in PVD neurons, we generated a strain that expressed sid-1 under the promoter mec-3, which exhibits strong expression in PVD. When Pmec-3::sid-1 is expressed in either nre-1(-)lin-15b(-) or lin-15b(-) backgrounds, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to the genetic background alone. Moreover, knockdown of genes playing role in dendrite regeneration in the nre-1(-)lin-15b(-), Pmec-3-sid-1[+] background resulted in significant reduction in dendrite regeneration following laser injury. The extent of dendrite regrowth due to the RNAi of aff-1 or ced-10 in our optimized strain was comparable to that of aff-1 and ced-10 mutants. Essentially, our strain expressing sid-1 in PVD neuron, provides an RNAi optimized platform for high throughput screening of genes involved in PVD development, maintenance and regeneration.


Introduction
The PVD neuron in C. elegans serves as an excellent model to understand the molecular basis of neuronal development and function [1].This neuron helps worm to process multiple sensory functions including harsh touch sensation and proprioception [2].Many concepts for neuronal development have been understood using PVD neuron as a model [3,4].PVD neuron displays a stereotypic structure characterized by an orthogonal array of dendritic branches that span a significant portion of worm's body [1,5].The higher order branches are arranged in a menorah like fashion [6] and the tertiary branches, from any given menorah and the adjacent menorahs, establish self-avoidance to prevent physical contact, facilitated by netrin unc-40 signaling [7][8][9].C. elegans specific fusogens AFF-1 and EFF-1 sculpt this elaborate dendritic architecture of PVD neuron [6].The guidance cue receptors and the F-actin cytoskeleton machineries collaborate to promote the extension of these branches [10,11].Furthermore, The quaternary branches are stabilized by the physical interactions with the epidermis and muscle through a molecular repertoire represented by these tissues [12,13].
The higher order branches of PVD undergo dynamic modulation in response to mechanical stimuli from the environment [14].Additionally, these dendrites display degeneration like phenomenon in old animals due to abnormal expression of immunological peptides [15].However, the molecular mechanisms governing these processes are poorly defined.It has also been seen that upon laser mediated injury, the primary dendrites exhibit various regeneration phenomena, including self-fusion between proximal and distal dendrites, branching, and regrowth [16][17][18].The self-fusion process is dependent on epidermal secretion of AFF-1 fusogen [17], while regrowth and branching rely on neuron specific function of CED-10 RAC GTPase [18].Recent reports have also described dendrite regeneration phenomena in both fly and vertebrate model systems [19][20][21].
A high throughput screening of conserved molecular pathways governing the dendrite regeneration and degeneration processes in PVD neuron might yield relevant insights into these processes.Since its discovery in the late 1990s, RNAi has been a powerful tool for gene silencing and functional genomic studies in C. elegans [22][23][24].This tool helps in an unbiased identification of the molecular pathways controlling a given biological process in worm system [25][26][27].However, it is often difficult to get an effective knockdown of genes in the neurons using the traditional feeding method [28].In order to overcome these limitations, researchers have identified various RNAi sensitive strains which exhibit an increased responsiveness to RNAi [29][30][31][32].For example, mutation in the tumor suppressor gene retinoblastoma (rb)/lin-15b enhances the p-granule formation in somatic cells, thereby promoting RNAi mediated gene silencing [31,33].Several reports have shown that tissue-specific over-expression of the transmembrane protein SID-1, which is a channel for transporting dsRNA, greatly facilitates the uptake of dsRNA by that tissue [34][35][36][37].This approach has been used to design strains sensitive for RNAi specifically in the motor neurons and touch neurons of C. elegans [34,38].
While there have been a few studies which utilize RNAi to knockdown candidate genes in PVD neurons, the penetrance of the associated neuronal phenotypes observed in these studies seems to be modest [7,39,40].This suggests that there might be a scope to enhance the efficacy of RNAi in PVD neurons through additional modifications to the existing RNAi sensitive strains.In this study, we systematically analyzed the effectiveness of RNAi in these sensitized backgrounds by assessing the loss of function phenotype of the genes known to regulate PVD dendrite morphology, such as mec-3, hpo-30 and tiam-1.We observed that the penetrance of the RNAi induced phenotype, for any of these genes in sid-1(-); lin-15b(-);Punc-119::sid-1[+] background is similar to the general sensitive background such as lin-1b(-) or nre-1(-)lin-15b (-).To improve the knockdown of these genes in PVD neurons specifically, we constructed a strain expressing the dsRNA channel SID-1 under the mec-3 promoter, which exhibits strong expression in PVD neurons.We found that this Pmec-3::sid-1 expression combined with RNAi sensitivity in either lin-15b(-) or nre-1(-)lin-15b(-) background, significantly enhances the penetrance of PVD neuron phenotypes associated with the knockdown of mec-3, hpo-30 and tiam-1 compared to the penetrance seen in lin-15b(-) or nre-1(-) lin-15b(-) backgrounds alone.We further showed that the extent of dendrite regeneration observed on RNAi mediated knockdown of ced-10 and aff-1 genes in nre-1(-) lin-15b(-); Pmec-3:: sid-1 [+] background is comparable to the loss of function mutant alleles of these genes.This illustrates that combining the general RNAi sensitivity of the nre-1(-) lin-15b(-) background with the PVD specific increase in sid-1 expression optimizes RNAi efficiency in PVD neurons.

RNAi mediated knockdown of PVD-specific genes in the existing RNAi sensitive backgrounds show mild phenotypes
The primary goal of this study was to identify a genetic background that can efficiently knockdown candidate genes in PVD neurons.In order to achieve this, we first aimed to optimize the conditions for induction of ds RNA production in the E. coli HT115 bacteria.This step was essential because different studies have used a wide range of induction conditions in their experiments [23,38,41].In our initial experiments we assessed the efficacy of knocking down of some ubiquitously expressing genes such as dhc-1 (dynein-heavy chain), unc-22 and gbp-1 in the wild-type N2 Bristol strain [41].In our hands, the induction of primary culture (condition-1) produced stronger phenotypes associated with the knockdown of these and a few other globally expressing genes in wild type strain compared to the induction of secondary culture (condition-2) (S1A Fig) .Therefore, we chose condition-1 to pursue RNAi for genes related to the nervous system (S1C Fig) .Our RNAi experiments on unc-14, unc-13, snb-1, unc-31, and unc-25 revealed that both the lin-15b(-) and sid-1(-); lin-15b(-); Punc-119::sid-1(+) strains produce consistent loss of function phenotypes of these neuronal genes (S1C Fig).
We next tested whether any of these strains can be used for effective knockdown of genes known to shape the architecture of PVD dendrites [5], thereby producing relevant phenotypes.The dendrites in PVD neuron span across the whole body (Fig 1A ) and show an orthogonal pattern in higher order branches (1 o , 2 o , 3 o & 4 o, in Fig 1A).We selected mec-3, hpo-30 and tiam-1 which are required cell autonomously for the formation of these higher order branches in PVD [5,42,43].We performed RNAi of mec-3 in lin-15b(-), nre-1(-)lin-15b(-) and sid-1(-); lin15b(-); Punc-119::sid-1(+) backgrounds and compared the dendritic arborization with the null mutant of mec-3 (Fig 1C -1E).In mec-3 (e1338) mutant, the secondary/primary ratio per unit length of primary is close to zero as there is no secondary branch in this mutant (Fig 1B and 1C) as seen before [43].Whereas the RNAi of mec-3 in the sensitive backgrounds caused a wide range of phenotypes (Fig 1B).Some animals had partial quaternary (P4) or complete absence of quaternary (P4 o +3 o +2 o +1 o or 3 o +2 o +1 o in Fig 1B), while in some, both quaternary and tertiary branches were missing (2 o +1 o in Fig 1B).In the rest of the population, the phenotype was like mec-3 null mutant, where all the higher order branches were missing (Fig 1B).Therefore, the phenotype related to the secondary/primary ratio in the sensitive backgrounds due to the RNAi of mec-3 gene was significantly weaker compared to the null mutant (Fig 1C).Likewise, both the quaternary/tertiary and tertiary/secondary ratios were significantly reduced in the sensitive backgrounds lin-15b(-) & nre-1(-)lin-15b(-) upon knockdown of mec-3 gene (Fig 1D and 1E).
These observations suggested that RNAi mediated knockdown of the candidate genes in the PVD neuron using available general and neuron specific RNAi sensitive strains is not as efficient as the loss of function mutations of respective genes.And this leaves a window open for further optimization of RNAi in PVD neurons to enhance its efficiency by making modifications to existing RNAi sensitive strains.

PVD specific expression of sid-1 helped knockdown of genes in PVD neuron
To enhance the phenotype associated to the RNAi of PVD-specific genes, we thought of expressing the dsRNA channel SID-1 using PVD specific promoter [35,36].The cell-specific expression of sid-1 in touch or motor neuron enhanced the phenotypes related to the knockdown of genes in these neuron [34,38].We screened for a promoter that strongly expresses in PVD neuron using the neuronal cell atlas (CENGEN) [45,46].We found that the expression of unc-119 gene in PVD neuron (103.59Transcripts per million/TPM) is relatively lower as compared to the expression level mec-3 in same neuron (4067.391TPM, CENGEN) [45].Additionally, mec-3 is expressed early in PVD, starting from early larval stages [7], allowing RNAi to begin early for targeted genes.Therefore, we generated an integrated strain that expresses sid-1 under the mec-3 promoter to test if it enhances RNAi in the PVD neurons.We made a single copy insertion of Pmec-3::sid-1[+] (Fig 2A -2C) by the mobilization of Mos1 transposon element [47,48].As a control experiment we expressed Pmec-3::GFP (Fig 2A ) using the same single copy insertion transgenic method [47].The strain expressing Pmec-3:: GFP showed GFP expression in PVD (Arrowhead, Fig 2B) as well as in the PLM and ALM neurons (Fig 2B ) validating the expression of mec-3 promoter in PVD neurons.Similarly we inserted Pmec-3::sid-1(+) cassette and tested this strain for enhanced RNAi by first assessing the RNAi against GFP in PVD neurons.As a control we used the transgenic strain, wdIs52, that expresses GFP under the promoter pF49H12.4,which is specific to PVD, AQR and the tail neuron.In the wild type wdIs52 background, RNAi against GFP causes loss of GFP expression in all these neurons(Fig 2E).However, the RNAi against GFP in the RNAi sensitive strain expressing Pmec-3::sid-1 caused exclusive loss of GFP reporter in PVD neuron (Fig 2D and  2E), confirming that effect of sid-1 is specific to PVD neuron.In 100% of the worms, in Pmec-3::sid-1 background, GFP was not visible in PVD neuron (Fig 2E).Further, to test for PVD specific phenotypic defects in the Pmec-3::sid-1 strain, we knocked down hpo-30 and tiam-1 using RNAi, We observed a significant reduction in density of quaternary branches as compared to knockdown in the wild-type worms (Fig 2F and 2G), indicating the PVD specific effectiveness of the sid-1 expression in the strain we developed.

The genes controlling dendrite regeneration pathways can be knocked down by RNAi in nre1(-)lin-15b(-); Pmec-3::sid-1[+] background
Since the RNAi mediated knockdown of mec-3, tiam-1 and hpo-30 produced strong phenotypes in nre1(-)lin-15b(-); Pmec-3::sid-1[+] strain, we were encouraged to test whether one can optimally use this background in dendrite regeneration studies.Previous work has shown that the primary dendrite of PVD upon laser injury shows regeneration response [16,18].The dendrite regeneration depends on the RAC GTPase CED-10, GEF TIAM-1 [18] and the fusogen molecule AFF-1 [17].Typically, following dendrotomy (Orange laser shots, Fig 4A ), the primary dendrite regrows (green traces in schematic Fig 4B and 4C) and reconnects (green arrowheads) to the distal end of the injured dendrites (Fig 4B and 4C).The tertiary branches corresponding to the menorahs of proximal and distal dendrites often are fused with each other (red highlighted boxes, Fig 4B ) to bypass the gap created due to the injury.This phenomenon is called menorah-menorah fusion [17].As reported before [18], we found that the extent of regrowth after dendrotomy, indicated by 'territory length', is significantly reduced in the loss of function mutant of ced-10(n3246) (Fig 4B and 4D).Similarly, the percentage of reconnection and menorah-menorah fusion events were also significantly reduced in ced-10 mutant (Fig 4E and 4F).Often there is a visible gap between the proximal and distal dendrites in the ced-10 mutant (blue arrowhead, Fig 4B).When we performed RNAi of ced-10 in nre1(-)lin-15b(-); Pmec-3::sid-1[+] strain, we noticed all of these phenomena (blue arrowhead, Fig 4C), as seen in the ced-10 mutant.There was a significant reduction in territory length, percentage of neurites showing reconnection and percentage of neurites showing menorah-menorah fusion, upon RNAi of ced-10 (Fig 4D -4F).More interestingly, the extent of reduction in the regeneration parameters was comparable in RNAi background and ced-10 and tiam-1 mutant (Fig 4D -4F).Similarly, the reduction in regeneration parameters due to the RNAi of aff-1 was also comparable to the aff-1 mutant (Fig 4B -4F).

Discussion
PVD neuron has been a great model to understand the development and function of nerve cells [1,2,6,50].Especially, the stereotypic and elaborate dendritic branches in PVD neuron make it an interesting system to explore the mechanism of dendrite development, maintenance, and regeneration.Combining genetics and cell biology, researchers have made progress in mechanistic understanding of how elaborate anatomy of dendrite in PVD neuron is developed [6,10].However, there is a big gap in our understanding of how the dendritic arbor is maintained in adulthood and how it is repaired after injury.
RNAi has been instrumental in identifying novel molecular pathways in nerve cell development [31] and neurite regeneration [51,52] using C. elegans.However, RNAi cannot phenocopy 100% the null-mutant phenotype for various reasons including the stability and availability of target mRNA, Moreover, RNAi in nervous system is highly variable depending on neuron type [53] and genes being targeted.Researchers have always had to try various sensitive strains in order to get effective knockdown of genes in neurons [7,31].The mutations in the genes such as lin-15b, nre-1 and eri-1, which negatively regulate RNAi process, are often used to enhance the phenotypes in neuron [7,[31][32][33].The lack of expression of the dsRNA channel SID-1 in the nervous system makes systemic RNAi inefficient in neuron [34].To overcome this challenge, researchers often mis-expressed sid-1 in neurons to enhance the RNAi efficiency [34,38].The existing strain that overexpresses sid-1 under pan-neuronal promoter Punc-119 did not give effective phenotype as compared to nre-1(-)lin-15b(-) or lin-15b(-) mutant alone for PVD specific phenotypes.Therefore, we ventured our effort to try stronger PVD-specific promoter for obtaining stronger phenotype.Our results with the knockdown of mec-3, hpo-30 and tiam-1 genes indicated that indeed addition of Pmec-3::sid-1 enhances the https://doi.org/10.1371/journal.pone.0298766.g004associated dendrite branching phenotypes in both the lin-15b(-) and nre-1(-)lin-15b(-) backgrounds.The nre-1(-) lin-15b(-) Pmec-3::sid-1(+) background also allows us to achieve the expected phenotype for dendrite regeneration following laser injury through the RNAi of ced-10 and aff-1.Therefore, this will be a highly useful tool for the researchers to study the questions related to PVD neuron.

C. elegans strains and genetics
In this study, C. elegans strains were maintained at 20˚C on E. coli OP50 bacterial lawn seeded over Nematode Growth Medium (NGM) plates [54].The loss of function mutation is represented as (-).For example, loss of function allele of mec-3 (e1338) represented as mec-3(-).The mutants used in this study are mostly loss of function by deletion or substitution unless otherwise mentioned.These mutants were obtained from Caenorhabditis Genetics Centre (CGC).The mutations crossed with wdIs52 [pF49H12.4::GFP] strain carrying PVD specific GFP marker to aid visualization and microscopy and genotyped using their respective primers.Details of strain used for the study is provided in S1 Table

Optimization of the induction of ds RNA expression
To optimize the induction of dsRNA expression in E. coli HT115 bacteria in our hand, we tried three different induction conditions suggested in previous reports [23,38,41] with some modification i.e. condition 1 (primary culture), condition 2 (secondary culture with IPTG induction), condition 3 (secondary culture without IPTG induction).After seeding the bacteria grown under each condition, L4 staged worms (5-10 worms) were transferred onto these NGM plates containing carbenicillin, tetracycline, IPTG and was allowed to grow and give progenies to conduct experiments.The bacteria expressing various dsRNA were obtained from Arhinger's and Vidal's Library [55,56] (S2 Table ).

Condition I
The E.coli HT115 bacteria carrying RNAi clones targeting specific genes were thawed from -80 deg and grown in Luria-Bertani (LB)-plates with 50 ug/ml carbenicillin and 12.5 ug/ml tetracycline and inoculated at 37˚C.Then a single colony was inoculated and grown at 37˚C in 4 ml LB in an incubator-shaker till it reached OD600 of 0.8.This primary culture was then pelleted and resuspended in 1X M9 buffer supplemented with 1.5 mM IPTG, carbenicillin and tetracycline.The resuspended culture was seeded onto NGM pates containing same concentration of carbenicillin, tetracycline, and IPTG.These plates were prepared two days in advance.Seeded plates were incubated at 25˚C for a duration of 36 hours for the induction of the RNAi construct within the bacteria.The condition-I involving the induction of primary culture was used before [41].

Condition II
The primary culture was grown in LB medium containing 50 ug/ml carbenicillin and 12.5 ug/ ml tetracycline at 37˚C overnight as described in 'condition I'.The overnight grown primary culture then used to set secondary culture at 1 in 4 dilution in LB media containing carbenicillin, tetracycline, and 1Mm IPTG.The secondary culture was kept at 37˚C incubator-shaker until it reached an OD600 of 0.5-0.6.Subsequently the bacteria was pelleted down and resuspended in LB medium containing IPTG, carbenicillin, and tetracycline.Resuspended bacteria was seeded onto NGM plates containing antibiotics and IPTG.The plates were kept for induction period of 8 hours at 25˚C.

Condition III
Same steps were performed as in Condition II with few modifications such as IPTG induction in secondary culture was not done and bacteria were grown until it reached OD600 of 0.5-0.6then,seeded plates were allowed to grow for 48hrs at room temperature as done before [38].

RNAi using optimized condition
First optimal condition for induction of RNAi in E. coli was determined by performing RNAi against genes whose knockdown is known to produce strong phenotypes such as failure embryo-hatching, sterility or twitching of muscle etc. in N2 wild type strain.For example, we tested dhc-1, gpb-1, unc-22 under three mentioned conditions in N2 background (S1A Fig) .In our hand, the Condition-I produced stronger phenotypes as compared to the Condition-II and Condition-III (S1A Fig) .Few other genes were also tested for further confirmation of the efficiency of 'Condition-I', such as par-1, par-3, skn-1, dnc-1, bir-1, pal-1, plk-1, ama-1, which are ubiquitously required in worm and RNAi of these genes produce global phenotype [41].RNAi of many of the tested genes resulted in 100% penetrance in the wild type N2 Bristol strain (S1B Fig) .Using this optimal induction condition (condition I), we performed RNAi of genes such as unc-14, unc-13, snb-1, unc-31, unc-25 that are pan-neuronally required [34,38], We used the strains that are shown to enhance RNAi sensitivity (S1C Fig) .For each respective gene (S1A-S1C Fig), ten P0s were fed RNAi and their progenies were scored after 3 days for phenotype.Therefore, the percentage of phenotypes was calculated from 300-350 progenies for phenotypes like uncoordinated, paralyzed, shrinker, and twitching.

Imaging of PVD neuron
The worms were mounted in 10 mM Levamisole hydrochloride (Sigma1) solution on the 5% agarose (Sigma1) pads made on the glass-slides.The worms were imaged with 63X/1.4NAoil objective of Nikon1 A1R confocal system at a voxel resolution of 0.41μm x 0.41μm x 1μm and tile imaging module using imaging lasers 488nm (GFP), 543nm (mCherry/RFP) with 1-1.8 AU pinhole at 512x512 pixel resolution files for further analysis.For regeneration study, images were obtained at 24 hrs post-injury using same imaging condition.

Dendrite branch quantification
PVD dendrite branch density was quantified as quaternary density, tertiary density and secondary density to normalize the phenotypes acquired in images (Fig 1B and 1F and 1I) encompassing from cell body till the middle of major dendrite using following formula: Quaternary density: Total number of quaternary dendrites / Total number of tertiary dendrites.Tertiary density: Total number of tertiary dendrites/ Total number of secondary dendrites.Secondary density: Total number of secondary dendrites / length of primary dendrite(μm).The length of primary dendrite was measured using Simple Neurite Tracer plugin in Fiji-ImageJ1.

Laser system and dendrotomy details
Dendrotomy were conducted on worms at the L4 stage using the Bruker1 ULTIMA system with spectraPhysics1 Two-photon femtosecond laser.This laser is tunable and operates in the infrared range (690-1040 nm).The laser output was controlled using Conoptics pockel cells.For visualization of the PVD and injury, lasers with wavelengths of 920nm and 720nm were used simultaneously by two sets of galvanometer mirror scanning X-Y [18].To prepare slides, worms were immobilized using Levamisole hydrochloride (10mM) on 5% agarose pads and mounted with Corning cover glass.Worm-containing slides were placed under 60X/0.9NAwater objective (Olympus1) with a pixel resolution of 0.29um x 0.29um.
During the experiments (Fig 4), the PVD dendrites were severed at the first branch point, approximately 10 microns away from the cell body, using the first laser shot.This was followed by one more consecutive shot with a relative distance of 10-15um from the previous shot, resulting visible gap.After injury, worms were transferred to freshly seeded NGM plates with E. coli OP50 or RNAi bacteria for further observation.

Dendrite regeneration quantification
Dendrite regeneration was quantified based on regrowth from site of injury and fusion related parameters like menorah-menorah fusion [16,17] where one big menorah is supported by more than one secondary branch (Fig 4B and 4C,red highlighted box).Also we see these regrowing neurites getting connected to distal dendrite (Fig 4B and 4C, green arrowhead) evaluated as reconnection events [18].The extent of territory covered by regrowing dendrite (Fig 4B and 4C, yellow dotted line) was measured using Simple Neurite Tracer plugin in Fiji-Ima-geJ1 tracking the longest regenerating dendrite from cell body to the end point.

Harsh touch behavior analysis
The worms fed on RNAi bacteria such as L4440 (empty vector/Control) and mec-3 (dsRNA against mec-3) were single-selfed in 10-20 plates using eye lash pick and left for few minutes.Videos were recorded while giving them harsh touch with platinum wire posterior to vulva, recording was done for nearly 20-30 seconds as described before [2,57].Percentage of worms with positive response were calculated which showed observable increase in speed indicating escape response after harsh touch as represented in Fig 3G.

Statistical analysis
The statistical analysis were performed using GraphPad Prism software (version 9.5.1).For two-sample comparisons, unpaired two tailed t-tests were used.When analyzing multiple samples, one way analysis of variance (ANOVA) was performed, followed by Tukey's multiple comparisons test.The data used for the ANOVA analysis consisted of naturally occurring data with a normal distribution spread.To compare population data, fraction values were calculated for each sample and compared using a two-tailed chi-square Fischer's exact contingency test.
The figure legends of the respective bar provide the information about the number of samples (n) and the number of biological replicates (N).The significance levels considered for all statistical experiments were p<0.033*, 0.002**, 0.001***.The details of statistical analysis for the all the plots associated with this study are available in S4 Table.
Fig 1G) as the quaternary branches are completely missing in this mutant (yellow arrow/ Fig 1F).The knockdown of hpo-30 using RNAi in the sensitive backgrounds also showed a reduction of quaternary branches (Fig 1F) although often some quaternary branches were still present (green arrow / Fig 1F)

Fig 1 .
Fig 1.Standard sensitive strains cannot efficiently knockdown genes in PVD to cause dendritic phenotype close to the respective loss of function mutant.A) The stitched confocal image of PVD neuron expressing GFP-reporter wdIs52 [pF49H12.4::GFP] shows the elaborate dendritic branches of this neuron.The magnified inset and its schematic indicate the PVD soma, hierarchy of dendritic branches (1˚/primary, 2˚/secondary, 3˚/Tertiary, and 4˚/ quaternary) and its ventral axon.(B), (F), (I) Confocal images (left) and its schematic tracings (right) show the range of phenotype for dendritic branches shown in different colors, caused due to RNAi against PVD-specific genes such as mec-3, hpo-30, tiam-1 in various sensitive background.The L4440 bacteria was used as RNAi control.The images of the respective loss of function mutants are also presented.For example, mec-3(-) represents the loss of function mutant for mec-3.The yellow arrows show the tertiary branch without any quaternary (3 o +2 o +1 o ).Similarly, the green arrows show the tertiary branches with partial presence of quaternary (P4 o +3 o +2 o +1 o .)The plots (C-E) shows the density of secondary, tertiary and quaternary dendrite to give the quantitative description of mec-3 RNAi and mutant phenotype.The plots (G-H) & (J-K) show the density of quaternary and tertiary dendrite due to the RNAi of hpo-30 and tiam-1.Scale bar is 25 μm for all the images in (B),(F),(I).Statistics: For (C-E),(G-H),(J-K) One-way ANOVA with Tukey's multiple comparison test, and number of worms (n), Biological replicates (N) are 15�n�81, 1�N�4, p<0.033*, 0.002**, 0.001***, ns, not significant.https://doi.org/10.1371/journal.pone.0298766.g001